Volume-regulated chloride channel regulates cell proliferation and is involved in the possible interaction between TMEM16A and LRRC8A in human metastatic oral squamous cell carcinoma cells
Volume-regulated chloride channel regulates cell proliferation and is involved in the possible interaction between TMEM16A and LRRC8A in human metastatic oral squamous cell carcinoma cells
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DOI：
10.1016/j.ejphar.2021.173881
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发表时间：
2021-02-06
影响因子：
5
通讯作者：
Morita, Hiromitsu
中科院分区：
文献类型：
--
作者：
Yoshimoto, Shohei;Matsuda, Miho;Morita, Hiromitsu
目的：容积调节阴离子通道（VRACs）在细胞容积调节中起重要作用。尽管在生理学上几乎是在世纪前定义的，但仅已知VRAC、TMEM 16 A、LRRC 8A和雌激素样肽-1（BEST 1）的分子候选物。本研究旨在探讨VRAC在口腔鳞癌细胞系HST-1中的功能意义。方法：采用细胞增殖实验、RT-PCR、Western blot和流式细胞术检测VRAC基因表达和细胞增殖的变化。采用膜片钳技术记录离子通道活性。结果：VRAC是一种低渗诱导的电流，与HST-1细胞的增殖有关，但与HaCaT细胞的增殖无关。VRAC在HST-1中的药理学特征与先前报道的相似。特异性VRAC抑制剂DCPIB可完全抑制HST-1细胞的VRAC和增殖，最终导致细胞凋亡。HST-1中的VRAC通过TMEM 16 A和LRRC 8A的敲低而减弱，而BEST 1的敲低影响细胞增殖。原位邻位连接实验表明，TMEM 16 A和LRRC 8A在等渗条件下（300 mOsM）共定位于细胞膜上，而在低渗条件下（250 mOsM）分离于细胞膜上。
Objectives: Volume-regulated anion channels (VRACs), expressed in various cells, play an important role in cell volume regulation. Despite being physiologically defined almost half a century ago, only the molecular candidates of VRAC, TMEM16A, LRRC8A, and bestrophin-1 (BEST1), are known. Here, we aimed to explore the functional significance of VRAC in, HST-1, an oral squamous cell carcinoma (OSCC) cell line.Methods: Cell proliferation assays, RT-PCR, Western blot, and flow cytometry were used to estimate changes in gene expression and cell proliferation. Ion channel activity was recorded using the patch-clamp technique. Specific genes were knocked-down by siRNA assays.Results: VRAC, identified as a hypotonicity-induced current, was highly functional and associated with the proliferation of HST-1 cells but not of HaCaT (a normal keratinocyte) cells. The pharmacological profile of VRAC in HST-1 was similar to that reported previously. DCPIB, a specific VRAC inhibitor, completely inhibited VRAC and proliferation of HST-1 cells, eventually leading to apoptosis. VRAC in HST-1 was attenuated by the knockdown of TMEM16A and LRRC8A, while knockdown of BEST1 affected cell proliferation. In situ proximity ligation assay showed that TMEM16A and LRRC8A co-localized under isotonic conditions (300 mOsM) but were separated under hypotonic conditions (250 mOsM) on the plasma membrane.Conclusions: We have found that VRAC acts to regulate the proliferation of human metastatic OSCC cells and the composition of VRAC may involve in the interactions between TMEM16A and LRRC8A in HST-1 cells.